Untangling the pro-fibrotic loop in pulmonary fibrosis: Synergy between substrate stiffness and soluble factors promotes alternative activation of macrophages

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Differential uptake of non-fouling particles by primary human neutrophils

The advent of targeted drug carriers has opened many new avenues for the delivery of therapeutics directly to the site of disease, reducing systemic side effects and enhancing the efficacy of therapeutic molecules. However, the packaging of therapeutics into particulate carriers for delivery comes with its own set of challenges and barriers. Among these, a great deal of research effort has focused on protecting carriers from clearance by phagocytes by altering carrier surface chemistry. Many groups have explored the use of polyethylene glycol (PEG) chain coatings to mitigate unwanted phagocytosis, as PEG is highly hydrophilic and is well-known for its anti-fouling propertiesNotably, very few papers have explored the effects of PEG on uptake by freshly obtained primary human phagocytes in physiological conditions, creating a disconnect between the prevailing literature and ultimate applications. In this work, we investigate the effect of PEGylation on uptake by primary human neutrophils in vitro, and compare these effects to several cell lines and other model phagocytic cells systems in evaluating the effects of surface chemistry on phagocytosis. We find that primary human neutrophils preferentially phagocytose PEGylated drug carriers, and that this effect is linked to factors present in human plasma. These findings have major implications for the efficacy of PEGylation in designing long-circulating drug carriers, as well as the need for thorough characterization of drug carrier platforms in a wide array of in vitro and in vivo assays. Please click Additional Files below to see the full abstract

Particle surface properties direct cellular immune responses in the lung

Nano- and micro-particulate carriers enable the site-specific delivery for controlled biological responses and can harness the intrinsic pathways by which the body responds to natural invaders. These particles are in the size range which naturally associates with many innate immune cells, including antigen presenting cells (APCs). Through controlled design properties, engineered nano- and microparticle drug delivery vehicles have the potential to expand the breadth of many therapeutic approaches, impacting immunological outcomes through cell-specific targeted delivery. However, in many applications, such as mucosal vaccines or controlled-release lung depots, optimal particle properties have not yet been identified. Physical properties such as size, shape, and surface chemistry are known to impact cellular interactions, particle margination, and biodistribution; as such, many particle design considerations have been established for systemic intravenous (IV) administration to create long-circulating drug delivery vehicles [3]. However, much less is known about particle design parameters which are critical to interfacing with and directing the immune system, especially through non-IV administration. Please click Additional Files below to see the full abstract

Engineered nanotherapeutics for pulmonary aerosol delivery

Despite centuries of use and widespread application, aerosol delivery of therapeutics remains limited to a small subset of diseases and active pharmaceutical ingredients, mainly restricted to small molecule delivery for asthma management. Respiratory diseases which would benefit from direct and localized treatment span a much larger landscape; chronic obstructive pulmonary disease (COPD), lower respiratory infections, and lung cancers alone globally contribute 7.8 million annual deaths, with a reported 117 million pulmonary cases (~37% of population, 2012) and over $88 billion in health care costs in the US[1, 2]. To expand the application of aerosol delivery, novel approaches are needed. To address this need, we have explored various applications of nanoparticle immune engineering for respiratory therapeutics[3]. Incorrect immune responses lie at the heart of most respiratory diseases and advances in these therapeutic areas requires consideration of the unique environment. Notably, the lung has an abundance of antigen presenting cells (APCs), such as macrophages and dendritic cells (DC), which phagocytose foreign materials at the air-lung interface. There are a number of lung-specific APC populations[4, 5]. Some subsets are well understood, however, other specialized subsets have only recently been identified due to historic challenges in differentiating these populations[6, 7]. Thus, there are many remaining questions as to the division of labor between these cells, their significance in different disease conditions, and their interactions with other adjacent cell populations at the mucosal interface[8]. Advancing this understanding is critical to develop new therapeutics; APCs are poised as the gatekeepers to lung regulation and lung DC-subset specifically are likely cellular targets for therapeutic intervention[9]. In order to better understand how these lung innate immune cells respond to inhaled particle therapeutics, we have developed sets of engineered particles with defined physical properties that originate at the molecular level. We have developed a series of metal organic framework (MOF) nanoparticle carriers with independently tunable particle size and internal porosity, enabling systematic investigation of the effect of particle pore structure on cellular interactions. These UIO-66 MOF derivatives have not only been optimized as pulmonary aerosol carriers but provide critical insight on the role of internal particle porosity following cellular internalization. To further modulate the lung immune environment and evaluate the role of ligand surface density on immune-modulation, we simultaneously developed a series of degradable polymeric nanoparticle carriers with controlled surface densities of two Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS), corresponding to TLR-4, and CpG oligodeoxynucleotide, corresponding to TLR-9[10]. Our in vitro results with murine bone marrow derived macrophages and in vivo studies following a direct instillation to murine airways both support a trade-off between particle dosage and optimal surface density; proinflammatory cytokine production was driven by the distribution of the adjuvant dose to a maximal number of innate cells, whereas the upregulation of costimulatory molecules on individual cells required an optimal density of TLR ligand on the particle surface. Taken together, results from these two sets of particle types demonstrate that both particle porosity and ligand surface density are critical parameters for tight control of immune stimulation and association with lung APCs and provide a foundation to build pathogen mimicking particle (PMP) vaccines and immunostimulatory therapeutics. References: 1. WHO: World Health Organization 2012. 2. NIH: National Heart, Lung, and Blood Institute 2012. 3. Moon, J. J.; Huang, B.; Irvine, D. J., Advanced materials (Deerfield Beach, Fla.) 2012, 24 (28), 3724-46. 4. Guilliams, M.; Lambrecht, B. N.; Hammad, H., Mucosal Immunol 2013, 6 (3), 464-73. 5. Kopf, M.; Schneider, C.; Nobs, S. P., Nat Immunol 2015, 16 (1), 36-44. 6. Blank, F.; Stumbles, P. A.; Seydoux, E.; Holt, P. G.; Fink, A.; Rothen-Rutishauser, B.; Strickland, D. H.; von Garnier, C., Am J Respir Cell Mol Biol 2013, 49 (1), 67-77. 7. Fytianos, K.; Drasler, B.; Blank, F.; von Garnier, C.; Seydoux, E.; Rodriguez-Lorenzo, L.; Petri-Fink, A.; Rothen-Rutishauser, B., Nanomedicine (Lond) 2016, 11 (18), 2457-2469. 8. Hasenberg, M.; Stegemann-Koniszewski, S.; Gunzer, M., Immunol Rev 2013, 25 (1), 189-214. 9. Zhao, L.; Seth, A.; Wibowo, N.; Zhao, C. X.; Mitter, N.; Yu, C.; Middelberg, A. P., Vaccine 2014, 32 (3), 327-37. 10. Noble, J.; Zimmerman, A.; Fromen, C. A., ACS Biomater Sci Eng 2017, 3 (4), 560-571

Experimental evaluation of receptor-ligand interactions of dual-targeted particles to inflamed endothelium

Vascular-targeted carriers (VTCs) are often designed as leukocyte mimics, conjugated with ligands that target leukocyte adhesion molecules (LAMs) to facilitate specific adhesion to diseased endothelium. VTCs must adhere in regions with dynamic blood flow, frequently requiring multiple ligand-receptor (LR) pairs to provide particle adhesion and high disease specificity. To study LR kinetics under flow, multiple research groups have used protein-coated plates to study the adhesion and rolling of dual-targeted particles in vitro.1-4 While important knowledge is contributed by these studies, they lack the complexity of a diseased physiologic endothelium, as spatiotemporal LAM expression varies widely. Despite decades of research with the ambition of mimicking leukocytes, the specificity of multiple LAM-targeted VTCs remains poorly understood, especially in physiological environments. More specifically, there is a lack of mechanistic understanding of how multiple ligands interact with biologically complex endothelial surfaces under dynamic in vivo environments. Please click Additional Files below to see the full abstract

Neue EASA-Anforderungen an Bodenverkehrsdienstleister – Chancen und Risiken für die Systempartner im Luftverkehr

The article “New EASA requirements for ground handling service providers – opportunities and risks for the various air transport system partners” identifies opportunities and risks arising from new requirements of the European Union Aviation Agency (EASA) for Ground Handling Service Providers (GHSP) from Rulemaking Task 0728. After summarizing these new requirements, an evaluation of expert interviews with representatives of ground handling service providers, airlines, airport operators, EASA and consulting firms is carried out using Mayring’s qualitative content analysis. The categorization is based on the distinction between opportunities and risks as well as the so-called PEST criteria (political, economic, social, technological). All stakeholders see both opportunities and risks associated with the new regulation, with the opportunities outweighing the risks only from the perspective of the aerodrome operators. Overall, the improvement in flight operational safety is identified as the greatest opportunity, while the potentially higher workload is identified as the main risk

Evaluation of receptorâ ligand mechanisms of dualâ targeted particles to an inflamed endothelium

Vascularâ targeted carriers (VTCs) are designed as leukocyte mimics, decorated with ligands that target leukocyte adhesion molecules (LAMs) and facilitate adhesion to diseased endothelium. VTCs require different design considerations than other targeted particle therapies; adhesion of VTCs in regions with dynamic blood flow requires multiple ligandâ receptor (LR) pairs that provide particle adhesion and disease specificity. Despite the ultimate goal of leukocyte mimicry, the specificity of multiple LAMâ targeted VTCs remains poorly understood, especially in physiological environments. Here, we investigate particle binding to an inflamed mesentery via intravital microscopy using a series of particles with wellâ controlled ligand properties. We find that the total number of sites of a single ligand can drive particle adhesion to the endothelium, however, combining ligands that target multiple LR pairs provides a more effective approach. Combining sites of sialyl Lewis A (sLeA) and antiâ intercellular adhesion moleculeâ 1 (aICAM), two adhesive molecules, resulted in â ¼3â 7â fold increase of adherent particles at the endothelium over singleâ ligand particles. At a constant total ligand density, a particle with a ratio of 75% sLeA: 25% aICAM resulted in more than 3â fold increase over all over other ligand ratios tested in our in vivo model. Combined with in vivo and in silico data, we find the best dualâ ligand design of a particle is heavily dependent on the surface expression of the endothelial cells, producing superior adhesion with more particle ligand for the lesserâ expressed receptor. These results establish the importance of considering LRâ kinetics in intelligent VTC ligand design for future therapeutics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/1/btm210008-sup-0007-suppinfo07.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/2/btm210008_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/133573/3/btm210008.pd

Computer modeling of aerosol diffusion through lung mucosa

Diseases of the lung are some of the most common and deadly in the world, accounting for 4 of the top 10 global causes of death according to the World Health Organization. Existing treatments, if any exist, tend to be extremely rigorous, invasive, and time-consuming. Further, due to the poor bioavailability of drugs traditionally administered orally or through injection, these treatments are not very effective. Technology is emerging that allows an aerosolized drug dosage to be delivered directly to the diseased area; however, the mucus layer separating the airways from the tissue (and the blood) remains a barrier to this method. In order to combat this, this research has constructed a physics-based computer model of the mucosal interface between the airways and the lung tissues, providing a much-needed insight into how a vaccine, antibiotic, or other drug must behave to effectively reach the target tissue in various lung regions.The lung has a phenomenal system called the mucociliary clearance mechanism in place to clear foreign particles (including cigarette ash, dust, and bacteria), prevent infection, and keep the lungs healthy. A layer of mucus on the surface of the inner lung is constantly pushed upward towards the throat by a bed of cilia, and most particles that impact on the mucus are cleared from the lungs quickly and without incident. However, when it fails to prevent a disease from being contracted, it remains a barrier to drug delivery, as those particles must cross the same thick mucus layer.The model uses COMSOL Multiphysics software to visualize the mucosa as a cross-section. Data from the literature is used to determine details like dimensions, velocity, and viscosity. The mucus layer moves upwards towards the throat in laminar flow, imitating the mucociliary effect, and the underlying periciliary layer has no net movement due to the regular beating of the cilia that move the mucus. The "drug" enters from the airway side and moves through the fluid by convection. Given these inputs, the model outputs an image showing how much, if any, of the administered particle diffuses through the mucosa and reaches the tissue. The model is extremely customizable, easily modified to simulate other drugs or any other particle so long as some properties are known. Variables have been specifically parametrized to find more complex relationships, like effective diffusivity, from an input of more readily available information, like particle radius. Lung conditions can also be quickly altered to meet the needs of the user (for example, the mucus layer is much thinner in the alveolar region, and the mucus of cystic fibrosis patients is much denser than average). Thus, the model can quickly provide greater insight into the efficacy of new lung treatments.Lew Wentz FoundationChemical Engineerin

Computer modeling of aerosol particle transport through lung mucosa

Diseases of the lung are some of the most common and deadly in the world, accounting for 4 of the top 10 global causes of death according to the World Health Organization. Due to the poor bioavailability of drugs traditionally administered orally or through injection, these treatments have limited efficacy for treating lung diseases. Technology is emerging that allows an aerosolized drug dosage to be delivered directly to the diseased area; however, the mucus layer separating the airways from the tissue (and the blood) remains a barrier to this method. In order to address this, we have constructed a physics-based computational fluid dynamics model of the mucosal interface between the airways and the lung tissues, providing insight into how a vaccine, antibiotic, or other drug must behave to effectively reach the target tissue in various lung regions.The lung has a system called the mucociliary clearance mechanism in place to clear foreign particles (e.g., cigarette ash, dust, and bacteria), prevent infection, and keep the lungs healthy. A layer of mucus on the surface of the inner lung is constantly pushed upward towards the throat by a bed of cilia, and most particles that impact on the mucus are cleared from the lungs quickly and without incident. This mechanism for preventing a disease from being contracted or from particulate damage remains a barrier to drug delivery, as those particles must cross the same thick, non-Newtonian mucus layer.We developed a model using COMSOL Multiphysics software to build a rectangular domain, simulating a cross-sectional slice of the radially symmetrical mucus layer. Data from the literature is used to determine details like mucus depth, velocity, and viscosity. The mucus layer moves upwards towards the throat in laminar flow, imitating the mucociliary effect, and the lower periciliary layer has slower net movement due to the regular beating of the cilia that move the mucus. The viscoelastic properties of mucus are accounted for in the model, with its shear-thinning effects parametrized to a Carreau model. The “drug” particles enter from the airway side and move through the fluid by convection. The Stokes-Einstein equation is used in conjunction with a hydrodynamic and steric hindrance model to calculate an effective diffusivity through the network of glycoproteins that comprises the mucus. Given these inputs, the model generates profiles showing particle concentrations at any point in the simulation domain at any particular time. The model is extremely customizable, easily modified to simulate other drugs or any other particle (including pathogens) provided some properties are known. Complex relationships may be calculated using a small number of easily-measured variables, such as particle diameter. Lung conditions can also be quickly altered to meet the needs of the user (for example, the mucus layer is much thinner in the alveolar region, and the mucus of cystic fibrosis patients is much denser than average). Thus, the model can quickly provide greater insight into the efficacy of new lung treatments, biomechanics of pathogens, and capacity of prophylactics. With the simulation results, we locate optimum dosage sites for a range of particle properties, as the advection of the mucus causes a maximized amount of drug to reach the epithelium some distance upstream of where it impacts the mucus. These results tell us how deep in the lungs a dosage must be delivered and how high its concentration must be to be efficacious.Lew Wentz FoundationChemical Engineerin

Nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells

AbstractEngineered nanoparticles have the potential to expand the breadth of pulmonary therapeutics, especially as respiratory vaccines. Notably, cationic nanoparticles have been demonstrated to produce superior local immune responses following pulmonary delivery; however, the cellular mechanisms of this increased response remain unknown. To this end, we investigated the cellular response of lung APCs following pulmonary instillation of anionic and cationic charged nanoparticles. While nanoparticles of both surface charges were capable of trafficking to the draining lymph node and were readily internalized by alveolar macrophages, both CD11b and CD103 lung dendritic cell (DC) subtypes preferentially associated with cationic nanoparticles. Instillation of cationic nanoparticles resulted in the upregulation of Ccl2 and Cxc10, which likely contributes to the recruitment of CD11b DCs to the lung. In total, these cellular mechanisms explain the increased efficacy of cationic formulations as a pulmonary vaccine carrier and provide critical benchmarks in the design of pulmonary vaccine nanoparticles.From the Clinical EditorAdvance in nanotechnology has allowed the production of precise nanoparticles as vaccines. In this regard, pulmonary delivery has the most potential. In this article, the authors investigated the interaction of nanoparticles with various types of lung antigen presenting cells in an attempt to understand the cellular mechanisms. The findings would further help the future design of much improved vaccines for clinical use